Previously, we developed a highly sensitive assay for viremia, capable of detecting a single copy of HIV-1 RNA in plasma. This sensitivity, capable of quantitating virions in plasma down to 0.3 copies RNA/ml or less, represents a 150-fold improvement over previous ultra-sensitive assays. In collaboration with colleagues at Abbott Laboratories, we have found that viremia in patients is independent of regimen, but strongly associated with pretherapy virus levels, implying that, even after 7 years of treatment, cells infected prior to therapy survive to make more virus. In collaboration with Dr. Robert Siliciano (Johns Hopkins Medical Institutions), we have obtained preliminary data demonstrating the lack of additional suppression of HIV-1 viremia on intensification of standard antiretroviral therapy with either protease inhibitor or NNRTI intensification. These studies are consistent with our recent findings (Maldarelli et al., PLoS Pathog. 2007; Palmer et al., PNAS 2008) indicating little or no active cycles of HIV-1 replication during standard drug therapy. These studies are being extended in clinical trials performed at NIH and elsewhere (Dr. Deborah McMahon, University of Pittsburgh) to ask what the decay rates of such cells are, and what happens to the levels if the treatment regimen is either simplified or intensified during therapy including using the new antiretroviral raltegravir to intensify therapy. We are also addressing the anatomic distribution of HIV-1 in infected patients prior to and following therapy. In collaboration with Dr. Phalguni Gupta (University of Pittsburgh), we are comparing the relative levels of HIV-1 in seminal and blood plasma in patients undergoing antiretroviral therapy. The results of these studies will provide important information directly relevant to the current controversy regarding the infectiousness of HIV-infected partners undergoing antiretroviral therapy. The same assay is being used (in collaboration with Dr. Bruce Walker, Harvard University) to probe the levels of viremia in the rare HIV-1-infected patients who are able to control their viremia at very low levels in the absence of therapy, as well as in a well-described NIH cohort of patients with HLA-restricted HIV-1 replication (with Drs. Stephen Migueles, Mark Conners, and H. Clifford Lane, NIAID) and also (in collaboration with Dr. David Margolis, University of North Carolina) to see whether a drug that activates HIV expression (i.e., valproic acid) can affect the level of persistent virus. As we investigate the nature of HIV-1 replication during suppressive therapy by investigating specific patient populations. In collaboration with Dr. Martin Markowitz (Rockefeller University), we are investigating the HIV-1 viremia in patients suppressed on antiretroviral therapy who initiated antiretroviral therapy during acute HIV seroconversion. Early therapy has been associated with blunted immune responses, and these studies will determine whether levels of viremia are lower than that detected in patients with chronic HIV-1 infection. In collaboration with Drs. Joseph Casazza and B. Graham (NIAID Vaccine Research Center), we are investigating the effect of experimental therapeutic DNA vaccination on viremia in chronically infected individuals suppressed on antiretroviral therapy. [Corresponds to Project 1 in the April 2007 site visit report of the Host-Virus Interaction Branch, HIV Drug Resistance Program]